Method for producing recombinant adenovirus

ABSTRACT

The invention concerns a method for producing recombinant adenovirus by which viral DNA is introduced in a packaging cell culture and the viruses produced are harvested after liberation in the supernatant. The invention also concerns the viruses produced and their use.

The present invention relates to a new process for the production ofrecombinant adenoviruses. It also relates to the purified viralpreparations produced according to this process.

Adenoviruses exhibit certain properties which are particularlyadvantageous for use as vector for the transfer of genes in genetherapy. In particular, they have a fairly broad host spectrum, arecapable of infecting quiescent cells, do not integrate into the genomeof the infected cell, and have not been associated, up until now, withmajor pathologies in man. Adenoviruses have thus been used to transfergenes of interest into the muscle (Ragot et al., Nature 361 (1993) 647),the liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervoussystem (Akli et al., Nature genetics 3 (1993) 224), and the like.

Adenoviruses are viruses with a linear double-stranded DNA having a sizeof about 36 (kilobases) kb. Their genome comprises especially aninverted repeat sequence (ITR) at each end, an encapsidation sequence(Psi), early genes and late genes. The principal early genes arecontained in the E1, E2, E3 and E4 regions. Among these, the genescontained in the E1 region in particular are necessary for viralpropagation. The principal late genes are contained in the L1 to L5regions. The genome of the Ad5 adenovirus has been completely sequencedand is accessible on a database (see especially Genebank M73260).Likewise, parts or even the whole of other adenoviral genomes (Ad2, Ad7,Ad12 and the like) have also been sequenced.

For their use in gene therapy, various vectors derived from adenoviruseshave been prepared, incorporating various therapeutic genes. In each ofthese constructs, the adenovirus was modified so as to render itincapable of replicating in the infected cell. Thus, the constructsdescribed in the prior art are adenoviruses from which the E1 region hasbeen deleted, which region is essential for the viral replication and atthe level of which the heterologous DNA sequences are inserted (Levreroet al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161).Moreover, to enhance the properties of the vector, it has been proposedto create other deletions or modifications in the adenovirus genome.Thus, a heat-sensitive point mutation was introduced into the ts125mutant, making it possible to inactivate the 72 kDa DNA binding protein(DBP) (Van der Vliet et al., 1975). Other vectors comprise a deletion ofanother region essential for the viral replication and/or propagation,the E4 region. The E4 region is indeed involved in the regulation of theexpression of the late genes, in the stability of the late nuclear RNAs,in the abolition of the expression of the host cell proteins and in theefficacy of replication of the viral DNA. Adenoviral vectors in whichthe E1 and E4 regions are deleted therefore possess a transcriptionalbackground noise and an expression of viral genes which are highlyreduced. Such vectors have been described for example in Applications WO94/28152, WO 95/02697, PCT/FR96/00088). In addition, vectors carrying amodification at the level of the IVa2 gene have also been described (WO96/10088).

The recombinant adenoviruses described in the literature are producedfrom different adenovirus serotypes. Indeed, various adenovirusserotypes exist whose structure and properties vary somewhat, but whichexhibit a comparable genetic organization. More particularly, therecombinant adenoviruses may be of human or animal origin. As regardsthe adenoviruses of human origin, there may be mentioned preferablythose classified in group C, in particular the adenoviruses of type 2(Ad2), 5 (Ad5), 7 (Ad7) or 12 (Ad12). Among the various adenoviruses ofanimal origin, there may be mentioned preferably the adenoviruses ofcanine origin, and especially all the strains of the CAV2 adenoviruses[Manhattan strain or A26/61 (ATCC VR-800) for example]. Otheradenoviruses of animal origin are cited especially in application WO94/26914 incorporated into the present by reference.

In a preferred embodiment of the invention, the recombinant adenovirusis a group C human adenovirus. More preferably, it is an Ad2 or Ad5adenovirus.

The recombinant adenoviruses are produced in an encapsidation line, thatis to say a cell line capable of complementing in trans one or morefunctions deficient in the recombinant adenoviral genome. One of theselines is for example the line 293 into which a portion of the adenovirusgenome has been integrated. More precisely, the line 293 is a humanembryonic kidney cell line containing the left end (about 11-12%) of theserotype 5 adenovirus (Ad5) genome, comprising the left ITR, theencapsidation region, the E1, including E1a and E1b, region, the regionencoding the pIX protein and a portion of the region encoding the pIVa2protein. This line is capable of transcomplementing recombinantadenoviruses defective for the E1 region, that is to say lacking all orpart of the E1 region, and of producing viral stocks having high titres.This line is also capable of producing, at a permissive temperature (32°C.), virus stocks comprising, in addition, the heat-sensitive E2mutation. Other cell lines capable of complementing the E1 region havebeen described, based especially on human lung carcinoma cells A549 (WO94/28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215).Moreover, the lines capable of transcomplementing several functions ofthe adenovirus have also been described. In particular, there may bementioned lines complementing the E1 and E4 regions (Yeh et al., J.Virol. 70 (1996) 559; Cancer Gen. Ther. 2 (1995) 322; Krougliak et al.,Hum. Gen. Ther. 6 (1995) 1575) and the lines complementing the E1 and E2regions (WO 94/28152, WO 95/02697, WO 95/27071).

Recombinant adenoviruses are usually produced by introducing viral DNAinto the encapsidation line, followed by lysis of the cells after about2 or 3 days (kinetics of the adenoviral cycle being from 24 to 36hours). After the lysis of the cells, the recombinant viral particlesare isolated by caesium chloride gradient centrifugation.

For the carrying out of the process, the viral DNA introduced may be thecomplete recombinant viral genome, optionally constructed in a bacterium(WO 96/25506) or in a yeast (WO 95/03400), transfected into the cells.It may also be a recombinant virus used to infect the encapsidationline. The viral DNA may also be introduced in the form of fragments eachcarrying a portion of the recombinant viral genome and a zone ofhomology allowing, after introduction into the encapsidation cell, theviral genome to be reconstituted by homologous recombination between thevarious fragments. A conventional process for the production ofadenoviruses thus comprises the following steps: the cells (for examplethe cells 293) are infected in a culture dish with a viral prestock inan amount of from 3 to 5 viral particles per cell (Multiplicity ofInfection (MOI)=3 to 5), or transfected with the viral DNA. Theincubation then lasts from 40 to 72 hours. The virus is then releasedfrom the nucleus by cell lysis, generally by several successive thawingcycles. The cellular lysate obtained is then centrifuged at low speed(2000 to 4000 rpm) and the supernatant (clarified cellular lysate) isthen purified by centrifugation in the presence of caesium chloride intwo steps:

-   -   A first rapid centrifugation of 1.5 hours on two caesium        chloride layers of densities 1.25 and 1.40 flanking the virus        density (1.34) so as to separate the virus from the proteins in        the medium;    -   A second longer gradient centrifugation (from 10 to 40 hours        depending on the rotor used), which constitutes the actual and        sole virus purification step.

Generally, after the second centrifugation step, the virus band ispredominant. Two fine, less dense bands are nevertheless observed whoseexamination by electron microscopy has shown that they are empty orbroken viral particles in the case of the more dense band, and viralsubunits (pentons, hexons) for the less dense band. After this step, thevirus is harvested by piercing, using a needle, in the centrifugationtube and the caesium is removed by dialysis or desalting.

Although the purity levels obtained are satisfactory, this type ofprocess has, nevertheless, certain disadvantages. In particular, it isbased on the use of caesium chloride, which is a reactive which isincompatible with a therapeutic use in man. Because of this, it isimperative to remove the caesium chloride at the end of thepurification. This process has, in addition, some other disadvantagesmentioned later, which limit its use on an industrial scale.

To overcome these problems, it has been proposed to purify the virusobtained after lysis, not using a caesium chloride gradient, but bychromatography. Thus, the article by Huyghe et al. (Hum. Gen. Ther. 6(1996) 1403) describes a study of different types of chromatographiesapplied to the purification of recombinant adenoviruses. This articledescribes especially a study of purification of recombinant adenovirusesusing a weak anion-exchange chromatography (DEAE). Previous studies havealready described the use of this type of chromatography for thispurpose (Klemperer et al., Virology 9 (1959) 536; Philipson L., Virology10 (1960) 459; Haruna et al., Virology 13 (1961) 264). The resultspresented in the article by Huyghe et al. show a fairly mediocreefficacy of the ion-exchange chromatography procedure recommended. Thus,the resolution obtained is average, the authors indicating that virusparticles are present in several chromatography peaks; the yield is low(viral particle yield: 67%; infectious particle yield: 49%); and theviral preparation obtained following this chromatographic step isimpure. In addition, a pretreatment of the virus with variousenzymes/proteins is necessary. This same article describes, moreover, astudy of the use of gel permeation chromatography, demonstrating a verypoor resolution and very low yields (15-20%).

The present invention describes a new process for the production ofrecombinant adenoviruses. The process according to the invention resultsfrom modifications of the previous processes at the level of theproduction phase and/or at the level of the purification phase. Theprocess according to the invention now makes it possible to obtain veryrapidly and in an industrializable manner virus stocks in a very highquantity and quality.

One of the first aspects of the invention relates more particularly to aprocess for the preparation of recombinant adenoviruses in which theviruses are harvested from the culture supernatant. Another aspect ofthe invention relates to a process for the preparation of adenovirusescomprising an ultrafiltration step. According to another aspect, theinvention also relates to a process for the purification of recombinantadenoviruses comprising an anion-exchange chromatography step. Thepresent invention also describes an improved purification process usinga gel permeation chromatography, optionally coupled to an anion-exchangechromatography. The process according to the invention makes it possibleto obtain viruses of high quality, in terms of purity, stability,morphology and infectivity, with very high yields and under productionconditions which are completely compatible with industrial requirementsand with the legislation relating to the production of therapeuticmolecules.

In particular, in terms of industrialization, the process of theinvention uses methods for the treatment of culture supernatants provenon a large scale for recombinant proteins, such as microfiltration ordepth filtration, and tangential ultrafiltration. Moreover, because ofthe stability of the virus at 37° C., this process allows a betterorganization at the industrial stage since, contrary to theintracellular method, the time of harvest does not need to be precise towithin a half-day. Furthermore, it ensures a maximum harvesting of thevirus, which is particularly important in the case of viruses defectivein several regions. Moreover, the process of the invention allows easierand more precise monitoring of the kinetics of production, directly onhomogeneous samples of supernatant, without pretreatment, allowingbetter reproducibility of the productions. The process according to theinvention also makes it possible to dispense with the cell lysis step.The cell lysis has several disadvantages. Thus, it may be difficult toenvisage breaking the cells by freeze-thaw cycles at the industriallevel. Moreover, the alternative methods of lysis (Dounce, X-press,sonication, mechanical shearing, and the like) have disadvantages: theyare potential generators of aerosols which are difficult to confine forthe L2 or L3 viruses (level of confinement of the viruses, dependent ontheir pathogenicity or on their mode of dissemination), these viruseshaving, moreover, a tendency to be infectious by the aerial route; theygenerate shear forces and/or a heat release which are difficult tocontrol, and which reduce the activity of the preparations. The solutionof using detergents to lyse the cells would need to be validated andwould require, in addition, validation of the removal of the detergent.Finally, the cell lysis leads to the presence, in the medium, ofnumerous cell debris which make the purification more complex. In termsof virus quality, the process of the invention potentially allows bettermaturation of the virus, leading to a more homogeneous population. Inparticular, since the packaging of the viral DNA is the last step of theviral cycle, the premature lysis of the cells potentially releases emptyparticles which, although non-replicative, are a priori infectious andcapable of taking part in the toxic effect specific to the virus and ofincreasing the specific activity ratio of the preparations obtained. Thespecific infectivity ratio of a preparation is defined as the ratio ofthe total number of viral particles, measured by biochemical methods(OD260 nm, CLHP, PCR, immunoenzymatic methods and the like) to thenumber of viral particles generating a biological effect (formation oflysis plaques on cells in culture in solid medium, transduction ofcells). In practice, for a purified preparation, this ratio isdetermined by calculating the ratio of the concentration of theparticles measured by OD at 260 nm to the concentration ofplaque-forming units of the preparation. This ratio should be less than100.

The results obtained show that the process of the invention makes itpossible to obtain a virus of a purity at least equal to its homologuepurified by caesium chloride gradient centrifugation, in a single isstep and without prior treatment, starting with a concentrated viralsupernatant.

A first object of the invention therefore relates to a process for theproduction of recombinant adenoviruses, characterized in that the viralDNA is introduced into a culture of encapsidation cells and the virusesproduced are harvested following release into the culture supernatant.Contrary to the prior processes in which the viruses are harvestedfollowing a premature cell lysis carried out mechanically or chemically,in the process of the invention, the cells are not lysed by means of anexternal factor. The culture is continued for a longer period, and theviruses are harvested directly in the supernatant, after simultaneousrelease by the encapsidation cells. The virus according to the inventionis thus recovered in the cell supernatant, whereas in the priorprocesses, it is an intracellular, more particularly internuclear,virus.

The Applicant has now shown that, in spite of the extension of theduration of the culture and in spite of the use of larger volumes, theprocess according to the invention makes it possible to generate viralparticles in large quantity and of better quality. In addition, asindicated above, this process makes it possible to avoid the lysis stepswhich are cumbersome at the industrial level and generate numerousimpurities.

The principle of the process is therefore based on the harvesting of theviruses released into the supernatant. This process may involve aculture time greater than that for prior techniques based on the lysisof the cells. As indicated above, the harvesting time does not have tobe precise within a half-day. It is essentially determined by thekinetics of release of the viruses into the culture supernatant.

The kinetics of release of the viruses may be monitored in various ways.In particular it is possible to use analytical methods such as RP-HPLC,IE-HPLC, semiquantitative PCR (Example 4.3), staining of dead cells withtrypan blue, measurement of the release of intracellular enzymes of theLDH type, measurement of the particles in the supernatant byCoulter-type apparatus or by diffraction of light, immunological methods(ELISA, RIA, and the like) or nephelometric methods, titration byaggregation in the presence of antibodies, and the like.

Preferably, the harvesting is carried out when at least 50% of theviruses have been released into the supernatant. The time when 50% ofthe viruses have been released may be easily determined by drawing akinetics according to the methods described above. Still morepreferably, the harvesting is carried out when at least 70% of theviruses have been released into the supernatant. It is particularlypreferable to carry out the harvesting when at least 90% of the viruseshave been released into the supernatant, that is to say when thekinetics reaches a plateau. The kinetics of release of the virus isessentially based on the adenovirus replication cycle and may beinfluenced by certain factors. In particular, it may vary according tothe type of virus used, and especially according to the type of deletionmade in the recombinant viral genome. In particular, the deletion of theE3 region appears to delay the release of the virus. Thus, in thepresence of the E3 region, the virus may be harvested from 24-48 hourspost-infection. In contrast, in the absence of the E3 region, a higherculture time appears to be necessary. In this regard, the Applicant hasperformed experiments on kinetics of release of an adenovirus deficientfor the E1 and E3 regions in the supernatant of the cells, and has shownthat the release starts about 4 to 5 days post-infection, and lasts upto day 14 approximately. The release generally reaches a plateau betweenday 8 and day 14, and the titre remains stable for at least 20 dayspost-infection.

Preferably, in the process of the invention, the cells are cultured fora period of between 2 and 14 days. Moreover, the release of the virusmay be induced by expression, in the encapsidation cell, of a protein,for example a viral protein, involved in the release of the virus. Thus,in the case of the adenovirus, the release may be modulated byexpression of the Death protein encoded by the E3 region of theadenovirus (protein E3-11.6K), optionally expressed under the control ofan inducible promoter. Because of this, it is possible to reduce thevirus release time and to harvest, in the culture supernatant, more than50% of the viruses 24-48 hours post-infection.

To recover the viral particles, the culture supernatant isadvantageously filtered beforehand. The adenovirus having a size ofabout 0.1 μm (120 nm), the filtration is performed by means of membraneshaving a porosity sufficiently large to allow the virus to pass through,but sufficiently fine to retain the contaminants. Preferably, thefiltration is carried out by means of membranes having a porositygreater than 0.2 μm. According to a particularly advantageousembodiment, the filtration is carried out by successive filtrations onmembranes of decreasing porosity. Particularly good results wereobtained by carrying out the filtration on depth filters of decreasingporosity 10 μm, 1.0 μm and 0.8-0.2 μm. According to another preferredvariant, the filtration is carried out by tangential microfiltration onflat membranes or hollow fibres. It is possible to use, moreparticularly, flat Millipore membranes or hollow fibres having aporosity of between 0.2 and 0.6 μm. The results presented in theexamples show that this filtration step has a yield of 100% (no loss ofvirus was observed by retention on the filter having the lowestporosity).

According to another aspect of the invention, the Applicant has nowdeveloped a process which makes it possible to harvest and purify thevirus from the supernatant. To this effect, the supernatant thusfiltered (or clarified) is subjected to ultra-filtration. Thisultrafiltration makes it possible (i) to concentrate the supernatant,the volumes involved being large, (ii) to carry out a first purificationof the virus and (iii) to adjust the preparation buffer to thesubsequent preparation steps. According to a preferred embodiment, thesupernatant is subjected to tangential ultrafiltration. Tangentialultrafiltration consists in concentrating and fractionating a solutionbetween two compartments, retentate and filtrate, separated by membraneswith a determined cut-off, by creating a flow in the retentatecompartment of the device and by applying a transmembrane pressurebetween this compartment and the filtrate compartment. The flow isgenerally produced by means of a pump in the retentate compartment ofthe device and the transmembrane pressure is controlled by means of avalve on the liquid stream of the retentate circuit or a variablecapacity pump on the liquid stream of the filtrate circuit. The speed offlow and the transmembrane pressure are chosen so as to generate lowshear forces (reynolds number less than 5000 sec⁻¹, preferably less than3000 sec⁻¹, pressure less than 1.0 bar) while avoiding blocking of themembranes. Various systems may be used to carry out the ultrafiltration,such as for example spiral membranes (Millipore, Amicon), flat membranesor hollow fibres (Amicon, Millipore, Sartorius, Pall, GF, Sepracor). Theadenovirus having a mass of about 1000 kDa, membranes having a cut-offof less than 1000 kDa, preferably of between 100 kDa and 1000 kDa, areadvantageously used within the framework of the invention. The use ofmembranes having a cut-off of 1000 kDa or greater indeed causes asubstantial loss of virus at this stage. Preferably, membranes having acut-off of between 200 and 600 kDa, still more preferably between 300and 500 kDa, are used. The experiments presented in the examples showthat the use of a membrane having a cut-off of 300 kDa allows theretention of more than 90% of the viral particles while removingcontaminants from the medium (DNA, proteins in the medium, cellularproteins and the like). The use of a cut-off of 500 kDa offers the sameadvantages.

The results presented in the Examples show that this step makes itpossible to concentrate large volumes of supernatant, without loss ofvirus (yield of 90%), and that it generates a virus of better quality.In particular, concentration factors of 20 to 100 fold can be easilyobtained.

This ultrafiltration step thus constitutes an additional purificationcompared with the conventional scheme since the contaminants with a massless than the cut-off (300 or 500 kDa) are removed, at least in part.The enhancement of the quality of the viral preparation is clear whenthe aspect of the separation is compared after the firstultracentrifugation step according to the two processes. In theconventional process involving lysis, the tube of viral preparation hasa cloudy appearance with a coagulum (lipids, proteins) which sometimestouches the virus band, whereas in the process of the invention, thepreparation after release and ultrafiltration has a band which isalready well resolved from the contaminants in the medium which persistin the top phase. The enhancement of the quality is also demonstratedwhen a comparison is made of the ion-exchange chromatography profiles ofa virus obtained by cell lysis relative to the virus obtained byultrafiltration as described in the present invention. Moreover, it ispossible to further enhance the quality by pursuing the ultrafiltrationby diafiltration of the concentrate. This diafiltration is carried outon the same principle as the tangential ultrafiltration, and makes itpossible to remove more completely the contaminants of size greater thanthe membrane cut-off, while equilibrating the concentrate in thepurification buffer.

Moreover, the Applicant has also shown that this ultrafiltration thenmakes it possible to purify the virus directly by chromatography on anion-exchange column or by gel permeation chromatography, making itpossible to obtain an excellent resolution of the viral particle peakwithout a need for treating the preparation prior to the chromatography.This is particularly unexpected and advantageous. Indeed, as indicatedin the article by Hyughe et al., cited above, the chromatographicpurification of viral preparations gives mediocre results and furtherrequires a pretreatment of the viral suspension with Benzonase andcyclodextrins.

More particularly, the process according to the invention is thereforecharacterized in that the viruses are harvested by ultrafiltration.

As indicated above, the resulting concentrate can be used directly forpurification of the virus. This purification may be carried out byprevious conventional techniques such as centrifugation on a caesiumchloride gradient or another ultracentrifugation medium allowing theparticles to be separated according to their size, density orsedimentation coefficient. The results presented in Example 4 show,indeed, that the virus thus obtained has remarkable characteristics. Inparticular, according to the invention, it is possible to replace thecaesium chloride with a solution of iodixanol,5,5′-[(2-hydroxy-1,3-propanediyl)bis(acetylimino)]-bis[N,N′-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamide]in which the virus sediments at equilibrium at a relative density ofbetween 1.16 and 1.24. The use of this solution is advantageous because,unlike caesium chloride, it is not toxic. Moreover, the Applicant hasalso shown that, advantageously, the concentrate obtained also makes itpossible to purify the virus directly by an ion-exchange mechanism or bygel permeation, and to obtain an excellent resolution of the viralparticle chromatographic peak without the need for pretreatment.

According to a preferred embodiment, the viruses are therefore harvestedand purified by anion-exchange chromatography.

For the anion-exchange chromatography, various types of supports may beused, such as cellulose, agarose (Sepharose gels), dextran (Sephadexgels), acrylamide (Sephacryl gels, Trisacryl gels), silica (TSK gels-SWgel), poly[styrene-divinylbenzene] (Source gels or Poros gels),ethyleneglycol-methacrylate copolymer (Toyopearl HW gels and TSK gels-PWgel), or mixtures (agarose-dextran: Superdex gel). Moreover, to enhancethe chromatographic resolution, it is preferable, within the frameworkof the invention, to use supports in the form of beads, having thefollowing characteristics:

-   -   as spherical as possible,    -   of calibrated diameter (beads which are all identical or which        are as homogeneous as possible), without imperfections or        breaks,    -   with the smallest possible diameter: beads of 10 μm have been        described (MonoBeads from Pharmacia or TSK gel from TosoHaas,        for example). This value appears to constitute the lower limit        for the diameter of beads whose porosity should, moreover, be        very high in order to allow penetration of the objects to be        chromatographed inside the beads (see below),    -   while remaining rigid in order to withstand pressure.

Moreover, to chromatograph the adenoviruses which constitute objects ofvery large size (diameter>100 nm), it is important to use gels having ahigh upper limit of porosity, or even as high as possible, so as toallow access of the viral particles to the functional groups with whichthey have to interact.

Advantageously, the support is chosen from agarose, dextran, acrylamide,silica, poly[styrene-divinylbenzene], ethyleneglycol-methacrylatecopolymer, alone or as a mixture.

For the anion-exchange chromatography, the support used should befunctionalized by grafting a group capable of interacting with ananionic molecule. Most generally, the group consists of an amine whichmay be ternary or quaternary. By using a ternary amine, such as DEAE forexample, a weak anion exchanger is obtained. By using a quaternaryamine, a strong anion exchanger is obtained.

Within the framework of the present invention, it is particularlyadvantageous to use a strong anion exchanger. Thus, a chromatographicsupport as indicated above, functionalized by quaternary amines, ispreferably used according to the invention. Among the supportsfunctionalized by quaternary amines, there may be mentioned, asexamples, the resins Source Q, Mono Q, Q Sepharose, Poros HQ and PorosQE, resins of the Fractogel TMAE type, and the Toyopearl Super Q resins.

Preferred examples of resins which can be used within the framework ofthe invention are the Source, especially Source Q, such as 15 Q(Pharmacia), the MonoBeads, such as Q (Pharmacia), the Poros HQ andPoros QE type resins. The MonoBeads support (diameter of the beads10±0.5 μm) has been commercially available for more than 10 years andthe resins of the Source (15 μm) or Poros (10 μm or 20 μm) type forabout 5 years. The latter two supports exhibit the advantage of having avery broad internal pore distribution (they range from 20 nm to 1 μm),thus allowing the passage of very large objects through the beads.Furthermore, they offer very little resistance to the circulation ofliquid through the gel (therefore very little pressure) and are veryrigid. The transport of solutes towards the functional groups with whichthey will interact is therefore very rapid. The Applicant has shown thatthese parameters are particularly important in the case of theadenovirus, whose diffusion is slow because of its size.

The results presented in the Examples show that the adenovirus may bepurified from the concentrate in a single anion-exchange chromatographystep, that the purification yield is excellent (140% in terms of tdu,compared with the value of 49% reported by Huyghes et al.) and that theresolution is excellent. In addition, the results presented show thatthe adenovirus obtained has a high infectivity, and therefore possessesthe characteristics required for a therapeutic use. Particularlyadvantageous results were obtained with a strong anion exchanger, thatis to say functionalized by quaternary amines, and especially with theresin Source Q. The resin Source Q15 is particularly preferred.

In this regard, another subject of the invention relates to a processfor the purification of recombinant adenoviruses from a biologicalmedium characterized in that it comprises a step of purification bystrong anion-exchange chromatography.

According to this variant, the biological medium may be a supernatant ofencapsidation cells producing the said virus, a lysate of encapsidationcells producing the said virus, or a prepurified solution of the saidvirus.

Preferably, the chromatography is carried out on a supportfunctionalized with a quaternary amine. Still according to a preferredmode, the support is chosen from agarose, dextran, acrylamide, silica,poly[styrene-divinylbenzene], ethyleneglycol-methacrylate copolymer,alone or as a mixture.

A particularly advantageous embodiment is characterized in that thechromatography is performed on a Source Q resin, preferably Q15.

Moreover, the process described above is advantageously carried outusing a supernatant of producing cells, and comprises a preliminaryultrafiltration step. This step is advantageously carried out under theconditions defined above, and in particular, it is a tangentialultrafiltration on a membrane having a cut-off of between 300 and 500kDa.

According to another embodiment of the process of the invention, theviruses are harvested and purified by gel permeation chromatography.

The gel permeation may be performed directly on the supernatant, on theconcentrate, or on the virus derived from the anion-exchangechromatography. The supports mentioned for the anion-exchangechromatography may be used in this step, but without functionalization.

In this regard, the preferred supports are agarose (Sepharose gels),dextran (Sephadex gels), acrylamide (Sephacryl gels, Trisacryl gels),silica (TSK gels-SW gel), ethyleneglycol-methacrylate copolymer(Toyopearl HW gels and TSK gels-PW gel), or mixtures (agarose-dextran:Superdex gel). Particularly preferred supports are:

-   -   Superdex 200HR (Pharmacia)    -   Sephacryl S-500HR, S-1000HR or S-2000 (Pharmacia)    -   TSK G6000 PW (TosoHaas).

A preferred process according to the invention therefore comprises anultrafiltration followed by an anion-exchange chromatography.

Another preferred process comprises an ultrafiltration followed by ananion-exchange chromatography, followed by a gel permeationchromatography.

Another variant of the invention relates to a process for thepurification of adenoviruses from a biological medium comprising a firststep of ultracentrifugation, a second step of dilution or dialysis, anda third step of anion-exchange chromatography. Preferably, according tothis variant, the first step is performed by rapid ultracentrifugationon a caesium chloride gradient. The term rapid means anultracentrifugation ranging from about 0.5 to 4 hours. During the secondstep, the virus is diluted or dialysed against buffer, in order tofacilitate its injection onto the chromatography gel, and theelimination of the ultracentrifugation medium. The third step isperformed using an anion, preferably strong anion, exchangechromatography as described above. In a typical experiment, startingwith the virus harvested in the supernatant (or optionallyintracellular), a 1st rapid ultracentrifugation is performed withcaesium chloride (as in Example 3). Next, after a simple dilution of thesample (for example with 10 volumes of buffer) or after a simpledialysis in buffer, the sample is subjected to ion-exchangechromatography (as in Example 5.1.). The advantage of this variant ofthe process of the invention comes from the fact that it uses 2 totallydifferent modes of separation of the virus (density and surface charge),which may possibly bring the virus to a level of quality combining theperformances of the 2 methods. In addition, the chromatography stepmakes it possible to remove simultaneously the medium used for theultracentrifugation (caesium chloride for example, or any otherequivalent medium mentioned above).

Another subject of the invention relates to the use of iodixanol,5,5′-[(2-hydroxy-1,3-propanediyl)bis(acetylimino)]bis[N,N′-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamide]for the purification of adenoviruses.

For carrying out the process of the invention, various adenovirusencapsidation cells may be used. In particular, the encapsidation cellsmay be prepared from various pharmaceutically utilizable cells, that isto say cultivatable under industrially acceptable conditions and nothaving a recognized pathogenic character. They may be established celllines or primary cultures and especially human retinoblasts, human lungcarcinoma cells, or embryonic kidney cells. They may be advantageouslycells of human origin which can be infected by an adenovirus. In thisregard, there may be mentioned the KB, Hela, 293, Vero, gmDBP6, HER,A549, HER cells and the like.

The cells of the KB line are derived from a human epidermal carcinoma.They are accessible at the ATCC (ref. CCL17) as well as the conditionsallowing their culture. The human cell line Hela is derived from a humanepithelium carcinoma. It is also accessible at the ATCC (ref. CCL2) aswell as the conditions allowing its culture. The line 293 cells arehuman embryonic kidney cells (Graham et al., J. Gen. Virol. 36 (1977)59). This line contains especially, integrated in its genome, the leftpart of the genome of the human adenovirus Ad5 (12%). The cell line gmDBP6 (Brough et al., Virology 190 (1992) 624) consists of Hela cellscarrying the adenovirus E2 gene under the control of the MMTV LTR.

They may also be cells of canine origin (BHK, MDCK, and the like). Inthis regard, the cells of the canine line MDCK are preferred. Theconditions for the culture of the MDCK cells have been describedespecially by Macatney et al., Science 44 (1988) 9.

Various encapsidation cell lines have been described in the literatureand are mentioned in the Examples. They are advantageously cells whichtranscomplement the adenovirus E1 function. Still more preferably, theyare cells which transcomplement the adenovirus E1 and E4 or E1 and E2afunctions. These cells are preferably derived from the human embryoniccells of the kidney or the retina, or human lung carcinomas.

The invention thus provides a process for the production of particularlyadvantageous recombinant adenoviruses. This process is suited to theproduction of recombinant viruses which are defective for one or moreregions, and in particular of viruses defective for the E1 region, orfor the E1 and E4 regions. Moreover, it is applicable to the productionof adenoviruses of various serotypes, as indicated above.

According to a particularly advantageous mode, the process of theinvention is used for the production of recombinant adenoviruses inwhich the E1 region is inactivated by deletion of a PvuII-BglII fragmentstretching from nucleotide 454 to nucleotide 3328, in the Ad5 adenovirussequence. This sequence is accessible in the literature and also on adatabase (see especially Genebank No. M73260). In another preferredembodiment, the E1 region is inactivated by deletion of an HinfII-Sau3Afragment stretching from nucleotide 382 to nucleotide 3446. In aspecific mode, the process allows the production of vectors comprising adeletion of the whole of the E4 region. This may be carried out byexcision of an MaeII-MscI fragment corresponding to nucleotides35835-32720. In another specific mode, only a functional part of E4 isdeleted. This part comprises at least the ORF3 and ORF6 frames. By wayof example, these coding frames can be deleted from the genome in theform of PvuII-AluI and BglII-PvuII fragments respectively, correspondingto nucleotides 34801-34329 and 34115-33126 respectively. The deletionsof the E4 region of the virus Ad2 dl808 or of the viruses Ad5 dl1004,Ad5 dl1007, Ad5 dl1011 or Ad5 dl1014 can also be used within theframework of the invention. In this regard, the cells of the inventionare particularly advantageous for the production of viruses comprisingan inactive E1 region and a deletion in the E4 region of the typepresent in the genome of Ad5 dl1014, that is to say of E4 virusesconserving the reading frame ORF4.

As indicated above, the deletion in the E1 region covers advantageouslyall or part of the E1A and E1B regions. This deletion should besufficient to render the virus incapable of autonomous replication in acell. The part of the E1 region which is deleted in the adenovirusesaccording to the invention advantageously covers nucleotides 454-3328 or382-3446.

The positions given above refer to the wild-type Ad5 adenovirus sequenceas published and accessible on a database. Although minor variations mayexist between the various adenovirus serotypes, these positions aregenerally applicable to the construction of recombinant adenovirusesaccording to the invention from any serotype, and especially theadenoviruses Ad2 and Ad7.

Moreover, the adenoviruses produced may possess other alterations intheir genome. In particular, other regions may be deleted in order toincrease the capacity of the virus and reduce its side effects linked tothe expression of viral genes. Thus, all or part of the E3 or IVa2region in particular may be deleted. As regards the E3 region, it mayhowever be particularly advantageous to conserve the part encoding thegp19K protein. This protein indeed makes it possible to prevent theadenoviral vector from becoming the subject of an immune reaction which(i) would limit its action and (ii) could have undesirable side effects.According to a specific mode, the E3 region is deleted and the sequenceencoding the gp19K protein is reintroduced under the control of aheterologous promoter.

As indicated above, adenoviruses constitute vectors for the transfer ofgenes which are very efficient for gene and cell therapy applications.For that, a heterologous nucleic acid sequence whose transfer and/orexpression into a cell, an organ or an organism is desired may beinserted into their genome. This sequence may contain one or moretherapeutic genes, such as a gene whose transcription and possibletranslation in the target cell generate products having a therapeuticeffect. Among the therapeutic products, there may be mentioned moreparticularly enzymes, blood derivatives, hormones, lymphokines:interleukins, interferons, TNF and the like (FR 9203120), growthfactors, neurotransmitters or their precursors or synthesis enzymes,trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5 and thelike; apolipoproteins: ApoAI, ApoAIV, ApoE and the like (WO 94/25073),dystrophin or a minidystrophin (WO 93/06223), tumour suppressor genes:p53, Rb, Rap1A, DCC, k-rev and the like (WO 94/24297), genes encodingfactors involved in coagulation: factors VII, VIII, IX and the like,suicide genes: thymidine kinase, cytosine deaminase and the like, oralternatively all or part of a natural or artificial immunoglobulin(Fab, ScFv and the like, WO 94/29446), and the like. The therapeuticgene may also be an antisense gene or sequence, whose expression in thetarget cell makes it possible to control the expression of genes or thetranscription of cellular mRNAs. Such sequences can for example betranscribed, in the target cell, into RNAs which are complementary tocellular mRNAs and can thus block their translation into protein,according to the technique described in Patent EP 140 308. Thetherapeutic gene may also be a gene encoding an antigenic peptide,capable of generating an immune response in man, for the production ofvaccines. They may be especially antigenic peptides specific for theEpstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573),the pseudo-rabies virus, or specific for tumours (EP 259 212).

Generally, the heterologous nucleic acid sequence also comprises atranscription promoter region which is functional in the infected cell,as well as a region situated in 3′ of the gene of interest, and whichspecifies a transcriptional end signal and a polyadenylation site. Allof these elements constitute the expression cassette. As regards thepromoter region, it may be a promoter region which is naturallyresponsible for the expression of the considered gene when the saidpromoter region is capable of functioning in the infected cell. It mayalso be regions of different origin (which are responsible for theexpression of other proteins, or which are even synthetic). Inparticular, they may be promoter sequences of eukaryotic or viral genesor any promoter or derived sequence, stimulating or repressing thetranscription of a gene in a specific manner or otherwise and in aninducible manner or otherwise. By way of example, they may be promotersequences derived from the genome of the cell which it is desired toinfect, or of the genome of a virus, especially the promoters of theadenovirus MLP, E1A genes, the RSV-LTR, CMV promoter, and the like.Among the eukaryotic promoters, there may also be mentioned theubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like),the promoters of the intermediate filaments (desmin, neurofilaments,keratin, GFAP and the like), the promoters of therapeutic genes (MDR,CFTR, factor VIII type and the like), the tissue-specific promoters(pyruvate kinase, villin, the promoter for the intestinal fattyacid-binding protein, the promoter for α-actin of the smooth musclecells, promoters specific for the liver; Apo AI, Apo AII, human albuminand the like) or alternatively the promoters which respond to a stimulus(steroid hormone receptor, retinoic acid receptor and the like). Inaddition these expression sequences may be modified by the addition ofactivating or regulatory sequences or of sequences allowing atissue-specific or predominant expression. Moreover, when the insertednucleic acid does not contain expression sequences, it may be insertedinto the genome of the defective virus downstream of such a sequence.

Moreover, the heterologous nucleic acid sequence may also contain, inparticular upstream of the therapeutic gene, a signal sequence directingthe synthesized therapeutic product in the secretory pathways of thetarget cell. This signal sequence may be the natural signal sequence forthe therapeutic product, but it may also be any other functionalsignal-sequence or an artificial signal sequence.

The expression cassette for the therapeutic gene may be inserted intovarious sites of the genome of the recombinant adenovirus, according tothe techniques described in the prior art. It can first of all beinserted at the level of the E1 deletion. It can also be inserted at thelevel of the E3 region, as an addition or as a substitution ofsequences. It can also be located at the level of the deleted E4 region.

The present invention also relates to the purified viral preparationsobtained according to the process of the invention, as well as anypharmaceutical composition comprising one or more defective recombinantadenoviruses prepared according to this process. The pharmaceuticalcompositions of the invention can be formulated for a topical, oral,parenteral, intranasal, intravenous, intramuscular, subcutaneous,intraocular or transdermal administration and the like.

Preferably, the pharmaceutical composition contains vehicles which arepharmaceutically acceptable for an injectable formulation. These may bein particular saline (monosodium or disodium phosphate, sodium,potassium, calcium or magnesium chloride and the like, or mixtures ofsuch salts), sterile or isotonic solutions, or dry, especiallyfreeze-dried, compositions which, upon addition depending on the case ofsterilized water or physiological saline, allow the constitution ofinjectable solutions. Other excipients can be used, such as, for examplea hydrogel. This hydrogel can be prepared from any biocompatible andnoncytotoxic polymer (homo or hetero). Such polymers have for examplebeen described in Application WO 93/08845. Some of them, such asespecially those obtained from ethylene and/or propylene oxide, arecommercially available. The virus doses used for the injection can beadjusted according to various parameters, and especially according tothe mode of administration used, the relevant pathology, the gene to beexpressed, or the desired duration of treatment. In general, therecombinant adenoviruses according to the invention are formulated andadministered in the form of doses of between 10⁴ and 10¹⁴ pfu, andpreferably 10⁶ to 10¹⁰ pfu. The term pfu (plaque forming unit)corresponds to the infectivity of an adenovirus solution, and isdetermined by infecting an appropriate cell culture and measuring,generally after 15 days, the number of infected cell plaques. Thetechniques for determining the pfu titre of a viral solution are welldocumented in the literature.

Depending on the therapeutic gene, the viruses thus produced can be usedfor the treatment or the prevention of numerous pathologies, includinggenetic diseases (dystrophy, cystic fibrosis and the like),neurodegenerative diseases (Alzheimer, Parkinson, ALS and the like),cancers, pathologies linked to coagulation disorders or todyslipoproteinaemias, pathologies linked to viral infections (hepatitis,AIDS and the like), and the like.

The present invention will be more fully described with the aid of thefollowing examples which should be considered as illustrative andnonlimiting.

LEGEND TO THE FIGURES

FIG. 1: Study of the stability of the adenovirus purified according toExample 4.

FIG. 2: HPLC (reversed phase) analysis of the adenovirus purifiedaccording to Example 4. Comparison with the adenovirus of Example 3.

FIG. 3: Kinetics of release of the adenovirus Ad-βGal in the supernatantof cells 293, measured by semiquantitative PCR and Plaque Assay.

FIG. 4: Elution profile on Source Q15 of an ultrafiltered adenovirussupernatant (Example 5.1).

FIG. 5: Ressource Q HPLC analysis of the virus peak harvested bychromatography on a Source Q15 resin of an ultrafiltered adenovirussupernatant (Example 5.1).

FIG. 6: (A) Elution profile on a Source Q15 resin of an ultrafilteredAd-APOA1 adenovirus supernatant (Example 5.3); and (B) HPLC (Resource Q)analysis of the virus peak harvested.

FIG. 7: (A) Elution profile on Source Q15 of an ultrafiltered Ad-TKadenovirus supernatant (Example 5.3). HPLC (Resource Q) analysis of thevarious virus fractions harvested (start and end of peak): (B) FractionF2, middle of the peak; (C) Fraction F3, limit of the peak; (D) FractionF4, end of the peak.

FIG. 8: Elution profile on a Mono Q resin of a concentrated supernatantof culture of adenovirus producing cells (Example 5.4). BG25F1:Supernatant virus concentrated and purified on caesium. BG25C:Concentrated infected supernatant.

FIG. 9: Elution profile on a POROS HQ gel of a concentrated supernatantof culture of adenovirus producing cells (Example 5.4). BG25F1: Virussupernatant concentrated and purified on caesium. BG25C: Concentratedinfected supernatant.

FIGS. 10 A and B: Gel permeation purification profile on SephacrylS1000HR/Superdex 200HR of an ultrafiltered adenovirus supernatant(Example 6).

FIG. 11: Analysis by electron microscopy of a stock of adenoviruspurified according to the invention.

FIG. 12: Analysis by electron microscopy of the virus band of density1.27.

GENERAL MOLECULAR BIOLOGY TECHNIQUES

The methods conventionally used in molecular biology, such aspreparative extractions of plasmid DNA, centrifugation of plasmid DNA incaesium chloride gradient, agarose or acrylamide gel electrophoresis,purification of DNA fragments by electroelution, phenol orphenol-chloroform extraction of proteins, ethanol or isopropanolprecipitation of DNA in saline medium, transformation in Escherichiacoli and the like, are well known to persons skilled in the art and arewidely described in the literature [Maniatis T. et al., “MolecularCloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1982; Ausubel F. M. et al., (eds), “CurrentProtocols in Molecular Biology”, John Wiley & Sons, New York, 1987].

The pBR322 and pUC type plasmids and the phages of the M13 series are ofcommercial origin (Bethesda Research Laboratories). For the ligations,the DNA fragments can be separated according to their size by agarose oracrylamide gel electrophoresis, extracted with phenol or with aphenol/chloroform mixture, precipitated with ethanol and then incubatedin the presence of phage T4 DNA ligase (Biolabs) according to therecommendations of the supplier. The filling of the protruding 5′ endscan be performed with the Klenow fragment of E. coli DNA polymerase I(Biolabs) according to the specifications of the supplier. Thedestruction of the protruding 3′ ends is performed in the presence ofphage T4 DNA polymerase (Biolabs) used according to the recommendationsof the manufacturer. The destruction of the protruding 5′ ends isperformed by a controlled treatment with S1 nuclease.

Site-directed mutagenesis in vitro by synthetic oligodeoxynucleotidescan be performed according to the method described by Taylor et al.,[Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed byAmersham. The enzymatic amplification of the DNA fragments by theso-called PCR technique [Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A.,Meth. Enzym. 155 (1987) 335-350] can be performed using a DNA thermalcycler (Perkin Elmer Cetus) according to the specifications of themanufacturer. The verification of the nucleotide sequences can beperformed by the method developed by Sanger et al., [Proc. Natl. Acad.Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.

EXAMPLES Example 1 Encapsidation Cell Lines

The encapsidation cells used within the framework of the invention maybe obtained from any cell line which can be infected by an adenovirusand which is compatible with a use for therapeutic purposes. They aremore preferably a cell chosen from the following lines:

The Cells of the 293 Line:

The 293 line is a human embryonic kidney cell line containing the leftend (about 11-12%) of the genome of the serotype 5 adenovirus (Ad5),comprising the left ITR, the encapsidation region, the E1 region,including E1a, E1b, the region encoding the pIX protein and a portion ofthe region encoding the pIVa2 protein (Graham et al., J. Gen. Virol. 36(1977) 59). This line is capable of transcomplementing recombinantadenoviruses defective for the E1 region, that is to say lacking all orpart of the E1 region, and of producing viral stocks having high titres.

The Cells of the A549 Line

Cells complementing the adenovirus E1 region were constructed from theA549 cells (Imler et al., Gene Ther. (1996) 75). These cells contain arestricted fragment of the E1 region, lacking the left ITR, placed underthe control of an inducible promoter.

The Cells of the HER Line

The human embryonic retinal (HER) cells may be infected with anadenovirus (Byrd et al., Oncogene 2 (1988) 477). Adenovirusencapsidation cells prepared from these cells have been described forexample in Application WO 94/28152 or in the article by Fallaux et al.(Hum. Gene Ther. (1996) 215). There may be mentioned more particularlythe line 911 comprising the E1 region of the Ad5 adenovirus genome, fromnucleotide 79 to nucleotide 5789 integrated into the genome of HERcells. This cell line allows the production of viruses defective for theE1 region.

The IGRP2 Cells

The IGRP2 cells are cells obtained from cells 293, by integration of afunctional unit of the E4 region under the control of an induciblepromoter. These cells allow the production of viruses defective for theE1 and E4 regions (Yeh et al., J. Virol (1996) 70).

The VK Cells

The VK cells (VK2-20 and VK10-9) are cells obtained from cells 293, byintegration of the entire E4 region under the control of an induciblepromoter, and of the region encoding the pIX protein. These cells allowthe production of viruses defective for the E1 and E4 regions (Krougliaket al., Hum. Gene Ther. 6 (1995) 1575).

The 293E4 Cells

The 293E4 cells are cells obtained from cells 293, by integration of theentire E4 region. These cells allow the production of viruses defectivefor the E1 and E4 regions (WO 95/02697; Cancer Gene Ther. (1995) 322).

Example 2 Viruses Used

The viruses produced in the context of the examples which follow are anadenovirus containing the E. coli LacZ marker gene (Ad-βGal), anadenovirus containing the gene encoding the type I herpes virusthymidine kinase (Ad-TK), an adenovirus containing the gene encoding thehuman p53 tumour suppressor protein and a virus encoding theapolipoprotein A1 (Ad-apoAI). These viruses are derived from the Ad5serotype and possess the following structure:

-   -   A deletion in the E1 region covering, for example, nucleotides        382 (HinfI site) to 3446 (Sau3a site).    -   A cassette for expression of the gene, under the control of the        RSV or CMV promoter, inserted at the level of the said deletion.    -   A deletion of the E3 region.

The construction of these viruses has been described in the literature(WO 94/25073, WO 95/14102, FR 95.01632, Stratford-Perricaudet et al. J.Clin. Invest (1992) p626). It is understood that any other construct maybe produced according to the process of the invention, and especiallyviruses carrying other heterologous genes and/or other deletions (E1/E4or E1/E2 for example).

Example 3 Production of Virus by Lysis of the Cells

This example reproduces the previous technique for producing viruses,consisting in lysing the encapsidation cells in order to recover theviruses produced.

The cells 293 are infected at 80-90% confluence in a culture dish with aprestock of Ad-βGal or Ad-TK virus (Example 2) in an amount of 3 to 5viruses per cell (Multiplicity of Infection MOI=3 to 5). The incubationlasts for 40 to 72 hours, the timing of the harvest being determined byobserving, under a microscope, cells which become round, become morerefringent and adhere increasingly weakly to the culture support. In theliterature, the kinetics of the viral cycle lasts for 24 to 36 hours.

At the level of the laboratory production, it is important to harvestthe cells before they become detached so as to remove the infectionmedium at the time of the harvest without losing cells and then to takethem up in a minimum volume (the concentration factor is, depending onthe size of the culture, of the order of 10 to 100 fold).

The virus is then released from the nucleus by 3 to 6 successive thawingcycles (ethanol-dry ice at −70° C., water bath at 37° C.).

The cell lysate obtained is then centrifuged at low speed (2000 to 4000rpm) and the supernatant (clarified cell lysate) is then purified byultracentrifugation on a caesium chloride gradient in two steps:

-   -   A first rapid ultracentrifugation (step) of 1.5 hours, 35000        rpm, rotor sw 41, on two caesium layers 1.25 and 1.40 flanking        the virus density (1.34) so as to separate the virus from the        proteins in the medium; the rotors may be “swinging” rotors        (Sw28, Sw41 Beckman) or fixed angle rotors (Ti 45, Ti 70, Ti        70.1 Beckman) depending on the volumes to be treated;    -   A second, longer gradient ultracentrifugation (from 10 to 40        hours depending on the rotor used), for example 18 hours at        35000 rpm in sw 41 rotor which constitutes the actual and sole        virus purification. The virus is present in a linear gradient at        equilibrium at a density of 1.34.

Generally, at this stage, the virus band is predominant. Nevertheless,two fine, less dense bands are sometimes observed whose examination byelectron microscopy has shown that they are empty or broken viruses andfor the least dense band, viral subunits (pentons, hexons). After thisstep, the virus is harvested in the tube by piercing with a needle andthe caesium is removed by dialysis or desalting on G25.

Example 4 Production of Virus in the Supernatant

This example describes an experiment for the production of virus byrecovering following spontaneous release. The virus is then harvested byultrafiltration and then purified by caesium chloride.

4.1. Procedure

In this method, unlike Example 3, the cells are not harvested 40 to 72hours post-infection, but the incubation is extended between 8 to 12days so as to obtain total lysis of the cells without the need to carryout the freeze-thaw cycles. The virus is present in the supernatant.

The supernatant is then clarified by filtration on depth filters ofdecreasing porosity (10 μm/1.0 μm/0.8-0.2 μm).

The virus has a size of 0.1 μm and at this stage, no loss of virus byretention on the filter at the lowest porosity (0.22 μm) was observed.

The supernatant, once clarified, is then concentrated by tangentialultrafiltration on a Millipore spiral membrane having a cut-off of 300kDa.

In the experiments reported in the present invention, the concentrationfactor is dictated by the dead volume of the system which is 100 ml.Supernatant volumes of 4 to 20 litres were concentrated with thissystem, making it possible to obtain concentrate volumes of 100 ml to200 ml without difficulty, which corresponds to a concentration factorof 20 to 100 fold.

The concentrate is then filtered on 0.22 μm and then purified bycentrifugation on caesium chloride as described in Example 3, followedby a dialysis step.

4.2 Results

Purity

Whereas the intracellular virus tube (Example 3) has a cloudy appearancewith a coagulum (lipids, proteins) which sometimes touches the virusband, the viral preparation obtained after the first centrifugation stepon caesium chloride by the process of the invention has a virus bandwhich is already well isolated from the contaminants in the medium whichpersist in the top phase. High-performance liquid chromatographyanalysis on a Resource Q column (cf Example 5) also shows this gain inthe purity of the starting material obtained by ultrafiltration ofinfected supernatant with a decrease in the nucleic acid contaminants(OD 260/280 ratio greater than or equal to 1.8) and protein contaminants(OD 260/280 ratio less than 1).

Stability of the Virus in a Supernatant at 37° C.:

The stability of the virus was determined by titration, by the plaqueassay method, of an infectious culture supernatant of which aliquotswere collected at various incubation times at 37° C. post-infection. Theresults are presented below:

Ad-TK Virus:

-   -   titre 10 days post-infection=3.5×10⁸ pfu/ml    -   titre 20 days post-infection=3.3×10⁸ pfu/ml Ad-βGal Virus    -   titre 8 days post-infection=5.8×10⁸ pfu/ml    -   titre 9 days post-infection=3.6×10⁸ pfu/ml    -   titre 10 days post-infection=3.5×10⁸ pfu/ml    -   titre 13 days post-infection=4.1×10⁸ pfu/ml    -   titre 16 days post-infection=5.5×10⁸ pfu/ml

The results obtained show that up to at least 20 days post-infection,the titre of the supernatant is stable within the limits of precision ofthe assay. Moreover, FIG. 1 shows that, in the elution buffer, the virusis stable for at least 8 months, at −80° C. and at −20° C.

Specific Infectivity of the Preparations

This parameter, corresponding to the ratio of the number of viralparticles measured by HPLC to the number of pfu, gives information onthe infectivity of the viral preparations. According to therecommendations of the FDA, it should be less than 100. This parameterwas measured as follows: Two series of culture flasks containing cells293 were infected in parallel at the same time with the same viralprestock under the same conditions. This experiment was carried out fora recombinant Ad-βgal adenovirus, and then repeated for an Ad-TKadenovirus. For each adenovirus, a series of flasks is harvested 48hours post-infection and is considered as a production of intracellularvirus purified on a caesium gradient after freezing-thawing. The otherseries is incubated for 10 days post-infection and the virus isharvested in the supernatant. The purified preparations obtained aretitrated by plaque assay and the quantification of the total number ofviral particles is determined by measuring the concentration of PVIIprotein by reversed phase HPLC on a C4 Vydac 4.6×50 mm column afterdenaturation of the samples in 6.4 M guanidine. The quantity of PVIIproteins is correlated to the number of viral particles considering 720copies of PVII per virus (Horwitz, Virology, second edition (1990)).This method is correlated with the measurements of viral particles onpreparations purified by the densitometric method at 260 nm, taking asspecific extinction coefficient 1.0 unit of absorbance=1.1×10¹²particles per ml.

The results obtained show that, for the Ad-βGal virus, this ratio is 16for the supernatant virus and 45 for the intracellular virus. For theAd-TK virus, the ratio is 78 in the case of the virus harvest in thesupernatant method and 80 for the virus harvested by the intracellularmethod.

Analysis by Electron Microscopy:

This method makes it possible to detect the presence of empty particlesor free viral subunits copurified, and to assess a protein contaminationof the purified viral preparations or the presence of non-dissociableaggregates of viral particles.

Procedure: 20 μl of sample are deposited on a carbon grid and thentreated for the examination by negative staining with 1.5% uranylacetate. For the examination, a 50 kV to 100 kv Jeol 1010 electronmicroscope is used.

Result: The analysis carried out on a virus harvested in the supernatantshows a clean preparation, without contaminants, without aggregates andwithout empty viral particles. It is furthermore possible to distinguishthe virus fibres as well as its regular geometric structure. Theseresults confirm the high quality of the viral particles obtainedaccording to the invention.

HPLC and SDS-PAGE Analysis of the Protein Profile:

SDS-PAGE Analysis:

20 μl of sample is diluted in Laemmli buffer (Nature 227 (1970)680-685), reduced for 5 min at 95° C., and then loaded onto Novex gels 1mm×16 wells gradient 4-20%. After migration, the gels are stained withcoomassie blue, and analysed on a Pharmacia VDS Image Master. Theanalysis reveals an electrophoretic profile for the virus harvested inthe supernatant in agreement with the literature data (LennartPhilipson, 1984 H. S. Ginsberg editors, Plenum press, 310-338).

Reversed Phase HPLC Analysis:

FIG. 2 shows the superposition of 3 chromatograms obtained from twovirus samples harvested intracellularly and a virus sample purified bythe supernatant method. The experimental conditions are the following:Vydac column ref. 254 Tp 5405, RPC4 4.6×50 mm, Solvent\A: H₂O+TFA 0.07%;Solvent B:CH3CN+TFA 0.07%, linear gradient: T=0 min % B=25; T-50 min %B=50%; flow rate=1 ml/min, detector=215 nm. The chromatograms show aperfect identity between the samples, without difference in the relativeintensities of each peak. The nature of each peak was determined bysequencing and shows that the proteins present are all of viral origin(see table below). PEAK (Min) IDENTIFICATION 19-20 Precursor PVII 21-22Precursor PVII; Precursor PX 1 to 12 27-28 Precursor PVI; Precursor PX32-33 Precursor PX 34 35-36 Mature PVII 37 Mature PVII; PVIII precursor39-41 Mature PVI 45 pX 46 pIX

Analysis In Vitro of the Efficiency of Transduction and of theCytotoxicity

The analysis of the cytotoxicity is carried out by infecting HCT116cells in 24-well plates for increasing MOIs and by determining thepercentage of live cells compared with a non-infected control, 2 and 5days post-infection, with the aid of the crystal violet stainingtechnique.

The results are presented in the table below: Adenovirus MOI = 3.0 MOI =10.0 MOI = 30.0 MOI = 100.0 Supernatant, D2 91% 96% 87% 89% Supernatant,D5 97% 90% 10% <5%

Analysis of the Transduction Efficiency

For an AD-βGal adenovirus, the transduction efficiency of a preparationis determined by infecting W162 cells, non-permissive to replication,cultured in 24-well plates, with increasing concentrations of viralparticles. For the same quantity of viral particles deposited, the cellsexpressing the beta-galactosidase activity are counted 48 hourspost-infection after incubation with X-gal as substrate. Each blue cellis counted as one transduction unit (TDU), the result is multiplied bythe dilution of the sample so as to obtain the concentration in units oftransduction of the sample. The transduction efficiency is thenexpressed by calculating the ratio of the concentration of viralparticles to the concentration in TDU. The results obtained show thatthe purified viruses have a good transduction efficiency in vitro.

Analysis of the Intracerebral Expression in Vivo

With the aim of evaluating the efficiency of the adenoviruses accordingto the invention for the transfer and expression of genes in vivo, theadenoviruses were injected by the stereotaxic route into the striatum ofOF1 immunocompetent mice. For that, volumes of 1 μl at 10⁷ pfu of viruswere injected at the following stereotaxic reference points (for theincision line at 0 mm): anteroposterior: +0.5; mediolateral: 2; depth:−3.5.

The brains were analysed 7 days after the injection. The resultsobtained show that the transduction efficiency is high: thousands oftransduced cells, very intense expression in the nucleus and frequentand intense diffusion in the cytoplasm.

4.3. Kinetics of Release of the Virus

This example describes a study of the kinetics of release ofadenoviruses in the encapsidation cell culture supernatant.

This study was carried out by semiquantitative PCR by means ofoligonucleotides complementary to the regions of the adenovirus genome.To this effect, the linearized viral DNA (1-10 ng) was incubated in thepresence of dXTP (2 μl, 10 mM), a pair of specific oligonucleotides andTaq polymerase (Cetus) in a 10×PCR buffer, and subjected to 30amplification cycles under the following conditions: 2 min at 91° C., 30cycles (1 min 91° C., 2 min at annealing temperature, 3 min at 72° C.),5 min 72° C., then 4° C. PCR experiments were carried out with the pairsof oligonucleotides of sequence: Pair 1: TAATTACCTGGGCGGCGAGCACGAT(6368) - SEQ ID No. 1 ACCTTGGATGGGACCGCTGGGAACA (6369) - SEQ ID No. 2Pair 2: TTTTTGATGCGTTTCTTACCTCTGG (6362) - SEQ ID No. 3CAGACAGCGATGCGGAAGAGAGTGA (6363) - SEQ ID No. 4 Pair 3:TGTTCCCAGCGGTCCCATCCAAGGT (6364) - SEQ ID No. 5AAGGACAAGCAGCCGAAGTAGAAGA (6365) - SEQ ID No. 6 Pair 4:GGATGATATGGTTGGACGCTGGAAG (6366) - SEQ ID No. 7AGGGCGGATGCQACGACACTGACTT (6367) - SEQ ID No. 8

The quantity of free adenovirus in the supernatant was determined on asupernatant of cells 293 infected with Ad-βGal, at various timespost-infection. The results obtained are presented in FIG. 3. They showthat the cellular release starts from the 5th or 6th day post-infection.

It is understood that any other virus determination technique may beused with the same objective, on any other encapsidation line, and forany adenovirus type.

Example 5 Purification of the Virus by Ultrafiltration and Ion Exchange

This example illustrates how the adenovirus contained in the concentratemay be purified directly and in a single ion-exchange chromatographystep, with very high yields.

5.1. Procedure

In this experiment, the starting material therefore consists of theconcentrate (or ultrafiltration retentate) described in Example 4. Thisretentate has a total protein content of between 5 and 50 mg/ml, andmore preferably between 10 and 30 mg/ml, in PBS buffer (10 mM phosphate,pH 7.2 containing 150 mM NaCl).

The ultrafiltration supernatant obtained from a virus preparation isinjected into a column containing Source Q 15 (Pharmacia) equilibratedin 50 mM Tris-HCl buffer pH 8.0 containing 250 mM NaCl, 1.0 mM MgCl₂ and10% glycerol (buffer A). After rinsing with 10 column volumes of bufferA, the adsorbed species are eluted with a linear NaCl gradient (250 mMto 1 M) on 25 column volumes at a linear flow rate of 60 to 300 cm/h,more preferably 12 cm/h. The typical elution profile obtained at 260 nmis presented in FIG. 4. The fraction containing the viral particles iscollected. It corresponds to a fine symmetrical peak whose retentiontime coincides with the retention time obtained with a preparation ofviral particles purified by ultracentrifugation. It is possible toinject, under the conditions described above, a minimum of 30 mg oftotal proteins per ml of Source Q 15 resin while preserving an excellentresolution of the viral particle peak.

In a representative experiment carried out using a β-gal adenoviruspreparation (Example 2), 12.6 mg of total proteins were injected onto aResource Q column (1 ml), that is to say 5×10¹⁰ PFU and 1.6×10¹⁰ TDU.The viral particle peak collected after chromatography (3.2 ml; FIG. 5)contained 173 μg of proteins and 3.2×10¹⁰ PFU and 2.3×10¹⁰ TDU. Theviral particles were therefore purified 70 fold (in terms of quantity ofproteins) and the purification yield is 64% in PFU and 142% in TDU (seetable below). Concentration Volumes Volumes Purification Steps sampledeposited recovered Yields factor SUPERNATANT 5000 ml 5000 ml 100% —ULTRA- Proteins: 6.3 mg/ml 5000 ml 200 ml 100% 5 FILTRATION PFU: 2.5 ×10¹⁰/ml 300 kd TDU: 8.1 × 10⁹/ml Particle 3.8 × 10¹¹/ml Part/pfu ratio:16.0 Part/tdu ratio: 47.0 ION-EXCHANGE PFU: 1.0 × 10¹⁰/ml 2.0 ml 3.0 mlProteins = 85% 70 PURIFICATION TDU: 7.2 × 10⁹/ml of concentrate ofelution PFU = 64% (one step) Particle: 2.0 × 10¹¹/ml TDU = 140% Part/pfuratio = 20 Particles = 84% Part/tdu ratio = 27 HPLC Purity = 98.4% CsClPFU: 1.0 × 10¹¹/ml 28.3 ml QSP 4.1 ml PFU = 66% 70 GRADIENT TDU: 7.5 ×10¹⁰/ml of concentrate TDU = 130% Particle: 2.2 × 10¹²/ml Particles =84% Part/pfu ratio = 22 HPLC Part/tdu ratio = 29 purity = 98.4%

5.2. Purity

After this purification step, the fraction collected has a purity≧98% ofviral particles (UV detection at 260 nm), when it is analysed byhigh-performance liquid chromatography (HPLC) on a Resource Q column (1ml) in the following chromatographic system: (10 μl of fraction purifiedby chromatography as described in Example 5.1 are injected into aResource Q15 column (1 ml of gel; Pharmacia) equilibrated in 50 mMTris/HCl buffer pH 8.0 (buffer B). After rinsing with 5 ml of buffer B,the adsorbed species are eluted with a linear gradient of 30 ml of NaCl(0 to 1 M) in the B buffer at a flow rate of 1 ml/min. The elutedspecies are detected at 260 nm. This HPLC analysis (FIG. 5) shows,furthermore, that the residual bovine serum albumin present in theultrafiltration retentate is completely removed during the preparativechromatography. Its content in the purified fraction is estimated to be<0.1%. Western blot analysis with an anti-BSA polyclonal antibody (withECL revealing; Amersham) indicates that the content of BSA in thechromatographic preparation is less than 100 ng per mg of virus.

The electrophoretic analysis of the adenoviral fraction purified bychromatography is performed on a polyacrylamide gel (4-20%) underdenaturing (SDS) conditions. The protein bands are then revealed withsilver nitrate. This analysis shows that the adenoviral preparationobtained by chromatography has a purity level at least equal to that ofthe preparation conventionally obtained by ultracentrifugation since ithas no additional protein band which would indicate a contamination ofthe preparation by non-adenoviral proteins.

The adenoviral preparation obtained by chromatography has an absorbanceratio A_(260 nm)/A_(280 nm) equal to 1.30±0.05. This value, which isidentical to that obtained for the best preparations obtained byultracentrifugation, indicates that the preparation is free ofcontaminating proteins or of contaminating nucleic acids.

Analysis by electron microscopy carried out under the conditionsdescribed in Example 4.2 on a chromatography-purified Ad-βgal virusshows a clean preparation, without contaminants, without aggregates andwithout empty viral particles (FIG. 11). Furthermore, caesium chloridegradient ultracentrifugation of this preparation reveals a single bandof density 1.30, which confirms the absence of contamination of thechromatographic preparations by potential empty particles or capsidfragments. During the purification, the chromatographic peak for thevirus is followed by a shoulder (or secondary peak) in its rear portion,which is not collected with the main peak. Caesium chloride gradientultracentrifugation of this fraction reveals a band of density 1.27, andanalysis of the composition of this fraction shows that it does notcontain nucleic acids. Analysis by electron microscopy shows that thisfraction contains particles of irregular shape, exhibiting perforationsat the surface (FIG. 12). They are therefore empty (lacking DNA) andincomplete particles. This therefore demonstrates that the purificationof the adenovirus by chromatography eliminates the empty particlespresent in a small quantity in the preparations before purification.

5.3. Purification of Adenoviruses Comprising a Therapeutic Gene Such asthe Genes Encoding the ApoA1 or Thymidine Kinase Proteins.

This example illustrates how adenoviruses comprising, in their genomes,heterologous nucleic acid sequences encoding therapeutic proteins may bepurified directly and in a single ion-exchange chromatography step. Italso shows that the chromatographic behaviour of the adenovirus isidentical to the heterologous nucleic acid sequences which it carries,allowing the same purification process to be used for differentadenoviruses carrying various heterologous nucleic acid sequences.

In a typical purification experiment, an adenovirus comprising, in itsgenome, a heterologous nucleic acid sequence encoding the ApoA1 protein(Example 2, WO 94/25073) is purified by chromatographing, in the systemdescribed in Example 5.1, 18 ml (72 mg of proteins; 1.08×10¹³ particles)of concentrated supernatant of a cell culture harvested 10 dayspost-infection (FIG. 6A). The viral particle peak collected afterchromatography (14 ml; 1.4 mg of proteins) contained 9.98×10¹²particles, which indicates a particle yield of 92% and a purificationfactor of 51. After this purification step, the fraction collected had(FIG. 6B) a purity greater than 98% as viral particles during thechromatographic analysis under the conditions described in 5.2. Theelectrophoretic analysis of the adenoviral fraction purified bychromatography carried out under the conditions described in Example 5.2showed that this preparation had a purity level at least equal to thatfor the preparation conventionally obtained by ultracentrifugation, andthat it is free of contaminating proteins or of contaminating nucleicacids.

In another typical purification experiment, an adenovirus comprising, inits genome, a heterologous nucleic acid sequence encoding the type 1herpes simplex thymidine kinase protein (Example 2, WO 95/14102) ispurified by chromatographing, in the system described in Example 5.1, 36ml (180 mg of proteins; 4.69×10¹³ particles) of concentrated supernatantof a cell culture (FIG. 7A). The viral particle peak collected afterchromatography (20 ml; 5.6 mg of proteins) contained 4.28×10¹³particles, which indicates a particle yield of 91% and a purificationfactor of 32. After this purification step, the fraction collected had(FIGS. 7B-D) a purity greater than 99% as viral particles during thechromatographic analysis under the conditions described in Example 5.2and an absorbance ratio of 1.29. The electrophoretic analysis of theadenoviral fraction purified by chromatography carried out under theconditions described in Example 5.2 showed that this preparation had apurity level at least equal to that of the preparation conventionallyobtained by ultracentrifugation, and that it is free of contaminatingproteins or of contaminating nucleic acids.

5.4. Purification of Intracellular Adenovirus by Strong Anion Exchange.

This example illustrates how an adenovirus comprising in its genome aheterologous nucleic acid sequence may be directly purified in a singleion-exchange chromatography step has from a lysate of encapsidationcells producing the said virus.

In a typical purification experiment, an adenovirus comprising in itsgenome a heterologous nucleic acid sequence encoding the β-gal proteinis purified by chromatographing in the system described in Example 5.1(FineLine Pilot 35 column, Pharmacia, 100 ml of Source 15 Q resin), 450ml (that is to say 2.5×10¹⁴ particles) of concentrated lysate of a cellculture harvested 3 days post-infection by chemical lysis (1% Tween-20).The viral particle peak collected after chromatography (110 ml)contained 2.15×10¹⁴ particles, which indicates a particle yield of 86%.After this purification step, the fraction collected had a viralparticle purity greater than 98% during the chromatographic analysisunder the conditions described in 5.2. Electrophoretic analysis of theadenoviral fraction purified by chromatography carried out under theconditions described in Example 5.2 showed that this preparation had alevel of purity at least equal has that of a preparation conventionallyobtained by ultracentrifugation, and that it lacks contaminatingproteins or contaminating nucleic acids.

5.5. Purification of the Virus by Ultrafiltration and Ion-ExchangeChromatography on Various Columns.

This example illustrates how the adenovirus contained in the concentratemay be directly purified and in a single ion-exchange chromatographystep using a gel different from the Source 15 Q support, while workingon the same separation principle, the anion exchange by interaction withthe quaternary amine groups of the matrix.

In a typical experiment for purification of the adenovirus, variousrecombinant adenoviruses (encoding βGal, apolipoprotein AI and TK) werepurified by chromatography on a Source Q30 gel column following theprotocol described in Example 5.1. The results obtained show that theSource Q30 gel makes it possible to obtain viral preparations of apurity of the order of 85%, with a yield of between 70 and 100%. Inaddition, the results obtained show that Q30 possesses, for thepurification of adenovirus, an efficiency (expressed by the number oftheoretical plates) of 1000 and a (maximum quantity of virus which maybe chromatographed without the peaks becoming altered) of 0.5 to 1×10¹²vp per ml. These results show that the Source Q30 gel may therefore besuitable for the purification of recombinant adenoviruses, even thoughits properties remain inferior to those of Source Q15 (purity of theorder of 99%, efficiency of the order of 8000 and capacity of the orderof 2.5 to 5×10¹² vp per ml).

In another typical adenovirus purification experiment, a β-Galadenovirus is purified by chromatography on a MonoQ HR 5/5 column usingthe procedure described in Example 5.1. The chromatographic imagecorresponding to the ultrafiltration retentate and to the purified viralpreparation thus obtained is illustrated in FIG. 8.

In another typical adenovirus purification experiment, a β-Galadenovirus is purified by chromatography on a Poros HQ/M columnaccording to the procedure described in Example 5.1. The chromatographicimage corresponding to the ultrafiltration retentate and to the purifiedviral preparation thus obtained is illustrated in FIG. 9.

Example 6 Purification of the Virus by Ultrafiltration and GelPermeation

This example illustrates how the adenovirus contained in the concentrate(ultrafiltration retentate) may be purified directly by gel permeationchromatography, with very high yields.

6.1. Procedure

200 μl of the ultrafiltration retentate obtained in Example 4 (that isto say 1.3 mg of proteins) are injected into an HR 10/30 column(Pharmacia) filled with Sephacryl S-1000SF (Pharmacia) equilibrated forexample in PBS buffer, pH 7.2 containing 150 mM NaCl (buffer C). Thespecies are fractionated and eluted with buffer C at a flow rate of 0.5ml/min and detected at the outlet of the column by UV at 260 nm.Alternatively, it is possible to use, under the same operatingconditions, a column filled with Sephacryl S-2000, which allows a betterresolution than the column Sephacryl S-1000HR for particles of 100 nm to1000 nm.

The resolution of the two gel permeation chromatographic systemsdescribed above may be advantageously enhanced by chromatographing theultrafiltration supernatant (200 μl) on a system of 2 HR 10/30 columns(Pharmacia) coupled in series (Sephacryl S-1000HR or S-2000 columnfollowed by a Superdex 200 HR column) equilibrated in the buffer C. Thespecies are eluted with the buffer C at a flow rate of 0.5 ml/min anddetected by UV at 260 nm. In this system, the viral particle peak isvery clearly better separated from the lower-molecular weight speciesthan in the system comprising a Sephacryl S-1000 HR or Sephacryl S-2000column alone.

In a representative experiment, an ultrafiltration retentate (200 μl,1.3 mg of proteins) was chromatographed on a system of 2 SephacrylS-1000HR-Superdex 200 HR 10/30 columns (FIG. 10). The chromatographicpeak containing the viral particles was collected. Its retention timecoincides with the retention time obtained with a preparation of viralparticles purified by ultracentrifugation. The viral particle peakcollected after chromatography (7 ml) contained 28 μg of proteins and3.5×10⁹ PFU. Its analysis by analytical ion-exchange chromatographyunder the conditions described in Example 5.2 shows the presence of acontaminating peak which is more strongly retained on the analyticalcolumn, whose surface area represents about 25% of the surface area ofthe viral peak. Its 260 nm/280 n=absorbance ratio which has a value of1.86 indicates that this contaminating peak corresponds to nucleicacids. The viral particles were therefore purified about 50 fold (interms of quantity of proteins) and the purification yield is 85% as PFU.

Alternatively, it is possible to chromatograph the preparations of viralparticles (ultrafiltrates or fractions at the outlet of ananion-exchange chromatography) on a TSK G6000 PW column (7.5×300 mm;TosoHaas) equilibrated in the buffer C. The species are eluted with thebuffer C at a flow rate of 0.5 ml/min and detected in UV at 260 nm.Likewise, it may be advantageous to increase the resolution of thechromatographic system, in particular to increase the separation of theviral particle peak from the lower-molecular weight species, bychromatographing the ultrafiltration supernatant (50 to 200 μl) on asystem of 2 columns coupled in series [TSK G6000 PW column (7.5×300 mm)followed by a Superdex 200 HR column] equilibrated in the buffer C. Thespecies are eluted with the buffer C at a flow rate of 0.5 ml/min anddetected in UV at 260 nm.

Example 7 Purification of the Virus by Ultrafiltration, Ion Exchange andGel Permeation

The viral particle fraction derived from the anion-exchangechromatography (Example 5) may be advantageously chromatographed in oneof the gel permeation chromatographic systems described above, forexample with the aim of further enhancing the level of purity of theviral particles, but also mainly with the aim of packaging the viralparticles in a medium compatible with or adapted to subsequent uses ofthe viral preparation (injection, . . . ).

1-32. (canceled)
 33. A purified adenovirus composition, wherein thecomposition is essentially free of contaminating proteins as determinedby its absorbance ratio A_(260 nm)/A_(280 nm), western blot, or HPLC.34. The purified adenovirus composition of claim 33, wherein thecomposition has an absorbance ratio A_(260 nm)/A_(280 nm) equal to1.30+/−0.05.
 35. The purified adenovirus composition of claim 33,wherein the composition is essentially free of contaminating bovineserum albumin (BSA).
 36. The purified adenovirus composition of claim35, wherein the contaminating BSA content in the composition is lessthan 100 ng per mg of virus as determined by western blot analysis withan anti-BSA polyclonal antibody.
 37. The purified adenovirus compositionof claim 35, wherein the contaminating BSA content in the composition isless than 0.1% as determined by HPLC.
 38. A purified adenoviruscomposition, wherein the composition is essentially free ofcontaminating nucleic acids as determined by its absorbance ratioA_(260 nm)/A_(280 nm) or by electrophoretic analysis.
 39. The purifiedadenovirus composition of claim 38, wherein the composition has anabsorbance ratio A_(260 nm)/A_(280 nm) equal to 1.30+/−0.05.
 40. Thepurified adenovirus composition of claim 38, wherein the composition isessentially free of contaminating nucleic acid as determined byelectorphoretic analysis.
 41. A purified adenovirus composition, whereinthe composition forms a single band of density 1.30 upon CsCl gradientultracentrifugation.
 42. The purified adenovirus composition of claim41, wherein the composition does not comprise empty virus particles. 43.The purified adenovirus composition of claim 41, wherein the compositiondoes not comprise capsid fragments.
 44. The purified adenoviruscomposition of claim 41, wherein the composition does not compriseaggregated virus particles.
 45. A purified adenovirus composition,wherein the composition is purified 70 fold in terms of quantity oftotal proteins when compared to the same composition before a one-stepion-exchange purification.
 46. A purified adenovirus composition,wherein the composition has a ratio of number of viral particlesmeasured by HPLC to the number of plaque-forming units (pfu) that isbetween 16 and
 78. 47. A purified recombinant adenovirus composition,wherein the adenovirus composition is produced by a process comprising:(a) introducing adenoviral DNA into a culture of encapsidation cells;and (b) harvesting adenoviruses following their release into the culturesupernatant without lysis of the encapsidation cells by an externalfactor.
 48. A purified adenovirus composition, wherein the adenoviruscomposition is produced by a process comprising: (a) obtaining abiological medium comprising adenovirus; (b) performing a singlechromatography step comprising anion-exchange chromatography on thebiological medium; and (c) recovering the adenovirus with an adenoviralparticle yield of at least 70% of the viral particles present in thebiological medium prior to chromatography.